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J. Biol. Chem., Vol. 279, Issue 38, 39886-39894, September 17, 2004

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In Vitro Identification and Characterization of an Early Complex Linking HIV-1 Genomic RNA Recognition and Pr55^Gag Multimerization^*

Ariel Roldan, Rodney S. Russell, Bruno Marchand, Matthias Götte¶, Chen Liang¶, and Mark A. Wainberg¶||

From the McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada and the Departments of ^¶Experimental Medicine and Microbiology & Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada

The minimal protein requirements that drive virus-like particle formation of human immunodeficiency virus type 1 (HIV-1) have been established. The C-terminal domain of capsid (CTD-CA) and nucleocapsid (NC) are the most important domains in a so-called minimal Gag protein (mGag). The CTD is essential for Gag oligomerization. NC is known to bind and encapsidate HIV-1 genomic RNA. The spacer peptide, SP1, located between CA and NC is important for the multimerization process, viral maturation and recognition of HIV-1 genomic RNA by NC. In this study, we show that NC in the context of an mGag protein binds HIV-1 genomic RNA with almost 10-fold higher affinity. The protein region encompassing the 11th -helix of CA and the proposed -helix in the CA/SP1 boundary region play important roles in this increased binding capacity. Furthermore, sequences downstream from stem loop 4 of the HIV-1 genomic RNA are also important for this RNA-protein interaction. In gel shift assays using purified mGag and a model RNA spanning the region from +223 to +506 of HIV-1 genomic RNA, we have identified an early complex (EC) formation between 2 proteins and 1 RNA molecule. This EC was not present in experiments performed with a mutant mGag protein, which contains a CTD dimerization mutation (M318A). These data suggest that the dimerization interface of the CTD plays an important role in EC formation, and, as a consequence, in RNA-protein association and multimerization. We propose a model for the RNA-protein interaction, based on previous results and those presented in this study.

Received for publication, May 20, 2004 , and in revised form, July 6, 2004.

^* This research was supported by the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: McGill AIDS Centre, Lady Davis Institute-Jewish General Hospital, 3755 Cote Ste-Catherine, Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8260; Fax: 514-340-7537; E-mail: mark.wainberg{at}mcgill.ca.

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